(1) Field of the Invention
The present invention relates to a diffusion gradient chamber system for determining the affects of various environments on microorganisms. In particular the present invention relates to a system which enables recording of the results of tests in dynamically changing environments.
(2) Description of Related Art
Microbial life in nature exists in gradients of light intensity, temperature, pH, viscosity, oxygen and other soluble nutrients. Microbes have evolved mechanisms to position themselves in regions of such gradients that favor their growth and/or survival. Numerous examples exist of these phenomena. For example, the chemoautotrophic sulfide oxidizing bacterium Beggiatoa can respond rapidly to vertical gradients of oxygen, light, and sulfide. This response allows it to position itself optimally in a microaerobicniche where it can oxidize sulfide diffusing up from anoxic zones. The chemotactic response itself has been studied extensively in Escherichia coli. It is well understood that E.coli cells move in a random walk consisting of runs and tumbles and that E.coli is capable of biasing its random walk by increasing its run time when swimming up a gradient of a chemoattractant or down a gradient of a repellent, the end result is more rapid migration toward an attractant or away from a repellent. The sharply defined "blooms" of purple and green sulfur bacteria in the thermocline region of stratified lakes are a well known example of such positioning and reflect the presence of chemo- and photosensory, locomotory, and buoyancy mechanisms in the cells.
Most systems employed for isolation, cultivation and study of microbes in the laboratory are homogeneous and do not sufficiently imitate the dynamic and diffusive character of natural habitats. Consequently, there is only limited understanding of the behavior of microbes confronted with multiple gradients of environmental cues. Indeed, it may well be that natural habitats hold a variety of organisms capable of growth only within a very narrow range of environmental conditions. Such "stenobiotic" organisms might constitute much of the untapped diversity believed to exist in nature and represent organisms that have eluded isolation with conventional, homogeneous culture systems. In addition, it is possible that the physiological responses of organisms growing in gradients of nutrients may differ from those growing in homogeneous culture.
One approach to mimicking the spatial and temporal heterogeneity of natural habitats is to use gel-stabilized media within which defined diffusion gradients may be imposed. Pioneers of this approach include Caldwell and Wimpenny (Caldwell, D. E. and Hirsch, P., Can. J. Microbiol. 19, 53-58 (1973); Caldwell, D. E., Lai, S. H., and Tiedje, J. M., Bull. Ecol. Res. Comm. (Stockholm) 17:151-158 (1973); Wimpenny, J. W. T. and Errol Jones, One-Dimensional Gel-Stabilized Model Systems. CRC Handbook of Laboratory Model Systems for Microbial Ecosystems., Vol II (Wimpenny, J. W. T., ed.) CRC Press, Boca Raton (1988); Wimpenny, J. W. T., P. Waters and A. Peters, Gel-Plate Methods in Microbiology. CRC Handbook of Laboratory Model Systems for Microbial Ecosystems., Vol. I (Wimpenny, J. W. T., ed.) CRC Press, Boca Raton (1988); and Thomas, L. V., and Wimpenny, J. W. T., Applied and Environmental Microbiology, p. 1991-1997 (June 1993)) and others who created 1- and 2-dimensional gradients and used these to study the growth and behavior of pure cultures of bacteria, as well as of microbial communities in situ in lake sediments. More recently, Nelson (Nelson, D. C., Revsbech, N. P., and Jorgensen, B. B., Applied and Environmental Microbiology, p. 161-168 (July 1986)) and co-workers characterized the microscale growth of Beggiatoa at the interface of opposing O.sub.2 and HS gradients in semisolid medium, and they also verified the ability of this bacterium to grow chemolithoautotrophically. See also Wolfaardt, G. M., et al., Applied and Environmental Microbiology, p. 2388-2396 (Aug. 1993).
The system of the present invention uses a diffusion gradient chamber in the form of a box with four (4) sides which allows up to four (4) variables to be studied by introducing them into a gel medium and studying the result of the gradients as described in an Abstract by Emerson, D. and Breznak, J. A., (Wood's Hole, American Society of Microbiology (Oct. 21-25, 1990)). A similar presentation appears in an Abstract by J. A. Breznak, Emerson, D., Worden, M., and Oriel, J. P., International Symposium on Microbial Ecology, Sep. 10, 1992. The system described was not capable of recording the results.
The problem is that it is difficult to visualize the microorganisms growing on the media and even more difficult to produce pictures of the microorganisms. There are also problems with the materials of construction which affect the results in use of the chamber.